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An androstane (17β-hydroxy-17α-picolyl-androst-5-en-3β-yl-acetate (derivative A)) cancer inhibitor was successfully captured in a carrier made of nano-sized hydroxyapatite (HAp) coated with chitosan-PLGA polymer blends (Ch-PLGA). In our previous studies, we demonstrated that it was convenient to use spherical HAp/Ch-PLGA carriers as vehicles to target the lungs following intravenous administration. In this study, we used emulsification and subsequent freeze-drying to load the spherical HAp/Ch-PLGA carriers with varying contents of the derivative A, in order to examine the selective toxicity towards cancerous/healthy lung cells. The XRD and FT-IR techniques confirmed the drug loading process, and the content of the poorly water soluble derivative A was estimated directly via the DSC technique. The particles were spherical in shape with the d 50 distribution varying between 167 and 231 nm, whereas the content of the derivative A ranged from 6.5 to 19.3 wt%. Cell-selective cytotoxicity was examined simultaneously on two cell lines: human lung carcinoma (A549 ATCC CCL 185) and human lung fibroblasts (MRC-5 ATCC CCL 171). All particles exhibited nearly three times larger cytotoxicity towards cancer cells (A549) than towards healthy cells (MRC5), where the particles with the derivative A content of 6.5 wt% allowed for the viability of healthy cells >80%. Ninety-six hours after the treatment of cells with particles with different contents of derivative A (after incubation and recovery), recovery was faster in damaged healthy cells than in cancerous cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Physicochemical characterization of steroid analogs (triazole, tetrazole, toluenesulfonylhydrazide, nitrile, dinitrile and dione) is considered to be a very important step in further drug selection. This study applies to the determination of lipophilicity of previously synthesized steroid derivatives using reversed-phase high-performance liquid chromatography (RP HPLC). Chemometric aspect of chromatographic lipophilicity is given throughout multiple linear regression (MLR) quantitative structure-retention relationships (QSRR) approach. Minimal inhibitory concentration (MIC) is determined for two steroid derivatives possessing antimicrobial activity against Staphylococcus aureus. Molecular docking study was performed in order to identify the compound with the most promising potential as human cytochrome P450 CYP17A1inhibitor. Identified 3β-hydroxyandrost-5-eno[16,17-d]-1,2,3-triazole (I.2.) could be recommended for further trials for anticancer drugs and subjected to the absorption, distribution, metabolism, excretion and toxicity (ADMET) evaluation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present paper deals with chromatographic lipophilicity determination of twenty-nine selected steroid derivatives using reversed-phase high-performance liquid chromatography (RP HPLC) combined with two mobile phase, acetonitrile-water and methanol-water. Chromatographic behavior of four groups (triazole and tetrazole, toluenesulfonylhydrazide, nitrile and dinitrile and dione) of selected steroid derivatives was studied. Investigated compounds were grouped using principal component analysis (PCA) according to their logk values for both mobile phases. Grouping was in the very good accordance with the polarity and lipophilicity of the investigated compounds. QSRR (quantitative structure-retention relationship) approach was used to model chromatographic lipophilicity behavior using molecular descriptors. Modeling was performed using linear regression (LR) and multiple linear regression (MLR) methods. The most influential molecular descriptors were lipophilicity descriptors that are important for molecules ability to pass through biological membranes and geometrical descriptors. All established LR-QSRR and MLR-QSRR models were statistically validated by standards, cross- and external validation parameters as well as with two graphical methods. According to all these assessments, MLR models were better for chromatographic lipophilicity prediction. It was shown that chromatographic systems with methanol-water were better for modeling of logk than systems with acetonitrile-water, as well as the systems that contained lower volume fractions of organic component in mobile phase. Modeling was performed in order to obtain lipophilicity profiles of investigated compounds as future drug candidates of biomedical importance., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In an earlier study we demonstrated that hydroxyapatite nanoparticles coated with chitosan-poly(d,l)-lactide-co-glycolide (HAp/Ch-PLGA) target lungs following their intravenous injection into mice. In this study we utilize an emulsification process and freeze drying to load the composite HAp/Ch-PLGA particles with 17β-hydroxy-17α-picolyl-androst-5-en-3β-yl-acetate (A), a chemotherapeutic derivative of androstane and a novel compound with a selective anticancer activity against lung cancer cells. 1 H NMR and 13 C NMR techniques confirmed the intact structure of the derivative A following its entrapment within HAp/Ch-PLGA particles. The thermogravimetric and differential thermal analyses coupled with mass spectrometry were used to assess the thermal degradation products and properties of A-loaded HAp/Ch-PLGA. The loading efficiency, as indicated by the comparison of enthalpies of phase transitions in pure A and A-loaded HAp/Ch-PLGA, equaled 7.47wt.%. The release of A from HAp/Ch-PLGA was sustained, neither exhibiting a burst release nor plateauing after three weeks. Atomic force microscopy and particle size distribution analyses were used to confirm that the particles were spherical with a uniform size distribution of d 50 =168nm. In vitro cytotoxicity testing of A-loaded HAp/Ch-PLGA using MTT and trypan blue dye exclusion assays demonstrated that the particles were cytotoxic to the A549 human lung carcinoma cell line (46±2%), while simultaneously preserving high viability (83±3%) of regular MRC5 human lung fibroblasts and causing no harm to primary mouse lung fibroblasts. In conclusion, composite A-loaded HAp/Ch-PLGA particles could be seen as promising drug delivery platforms for selective cancer therapies, targeting malignant cells for destruction, while having a significantly lesser cytotoxic effect on the healthy cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Biological investigation was conducted to study in vitro antiproliferative and pro-apoptotic potential of selected 17α-picolyl and 17(E)-picolinylidene androstane derivatives. The antiproliferative impact was examined on six human tumor cell lines, including two types of breast (MCF-7 and MDA-MB-231), prostate (PC3), cervical (HeLa), colon (HT 29) and lung cancer (A549), as well as one normal fetal lung fibroblasts cell line (MRC-5). All derivatives selectively decreased proliferation of estrogen receptor negative MDA-MB-231 breast cancer cells after 48 h and 72 h treatment and compounds showed time-dependent activity. We used this cell line to investigate cell cycle modulation and apoptotic cell death induction by flow cytometry, expression of apoptotic proteins by Western blot and apoptotic morphology by visual observation. Tested androstane derivatives affected the cell cycle distribution and induced apoptosis and necrosis. Compounds had different and specific mode of action, depending on derivative type and exposure time. Some compounds induced significant apoptosis measured by Annexin V test compared to reference compound formestane. Higher expression of pro-apoptotic BAX, downregulation of anti-apoptotic Bcl-2 and cleavage of PARP protein were confirmed in almost all treated samples, but the lack of caspase-3 activation suggested the induction of apoptosis in caspase-independent manner. More cells with apoptotic morphology were observed in samples after prolonged treatment. Structure-activity relationship analysis was performed to find correlations between the structure variations of investigated derivatives and observed biological effects. Results of this study showed that some of the investigated androstane derivatives have good biomedical potential and could be candidates for anticancer drug development., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The heterocyclic ring at C-17 position of the androstane compounds plays an important role in biological activity. The aim of the present study was to synthesize and evaluate potential antitumor activity of different A-modified 17α-picolyl and 17(E)-picolinylidene androstane derivatives. In several synthetic steps, novel derivatives bearing the hydroximino, nitrile or lactame functions in A-ring were synthesized and characterized according to the spectral data, by mass analysis as well as XRD analysis (compounds 6, 13 and 15). The structurally most promising compounds 6, 11-17 were investigated as antitumor agents. The in vitro antiproliferative activity was evaluated against six human cancer cell lines: estrogen receptor negative (ER-) breast adenocarcinoma (MDA-MB-231); estrogen receptor positive (ER+) breast adenocarcinoma (MCF-7); prostate cancer (PC-3); human cervical carcinoma (HeLa); lung adenocarcinoma (A549) and colon adenocarcinoma (HT-29) using MTT assay. The results of the 48h incubation time in vitro tests showed that compound 15 was the most effective against PC-3 (IC50 6.6μM), compound 17 against MCF-7 (IC50 7.9μM) cells, while compound 16 exhibited strong antiproliferative effect against both, MCF-7 (IC50 1.7μM) and PC-3 (IC50 8.7μM) cancer cells. It was also found that compounds 16 and 17 induced apoptosis in MCF-7 cells (dicyano derivative 17 stronger then dioxime 16 and reference formestane), with no distinct changes in the cell cycle of MCF-7 cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

17α-hydroxylase-C17,20-lyase (P45017α) is a key regulator enzyme of the steroid hormone biosynthesis in both the adrenals and the testes. Inhibition of this enzyme can block androgen synthesis in an early step, and may thereby be useful in the treatment of several androgen-dependent diseases. We developed radio-substrate in vitro incubation methods for the determination of the distinct 17α-hydroxylase and C17,20-lyase activities of the enzyme using rat testicular homogenate as enzyme source. With this method we have studied the inhibiting activity of selected steroidal picolyl and picolinylidene compounds. Tests revealed a substantial inhibitory action of the 17-picolinyliden-androst-4-en-3-one compound.

We report the synthesis of steroidal 16,17-seco-16,17a-dinitriles and investigate their antitumor cell properties. Compounds were evaluated for anticancer potential by in vitro antiproliferation studies, molecular docking and virtual screening. Several compounds inhibit the growth of breast and prostate cancer cell lines (MCF-7, MDA-MB-231 and PC3), and/or cervical cancer cells (HeLa). Supporting this, molecular docking predicts that steroidal 16,17-seco-16,17a-dinitriles could bind with high affinity to multiple molecular targets of breast and prostate cancer treatment (aromatase, estrogen receptor α, androgen receptor and 17α-hydroxylase) facilitated by D-seco flexibility and nitrile-mediated contacts. Thus, 16,17-seco-16,17a-dinitriles may be useful for the design of inhibitors of multiple steroidogenesis pathways. Strikingly, 10, a 1,4-dien-3-on derivative, displayed selective submicromolar antiproliferative activity against hormone-dependent (MCF-7) and -independent (MDA-MB-231) breast cancer cells (IC50 0.52, 0.11μM, respectively). Ligand-based 3D similarity searches suggest AKR1C, 17β-HSD and/or 3β-HSD subfamilies as responsible for this antiproliferative activity, while fast molecular docking identified AKR1C and ERβ as potential binders-both targets in the treatment of hormone-independent breast cancers., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A convenient microwave assisted solvent free synthesis as well as conventional synthesis of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives 7-19 from appropriate steroidal precursors 1-6 and methyl salicylate is reported. The microwave assisted synthesis in most cases was more successful regarding reaction time and product yields. It was more environmentally friendly too, compared to the conventional method. The antioxidant activity and cytotoxicity of the synthesized derivatives were evaluated in a series of in vitro tests, as well as their inhibition potency exerted on hydroxysteroid dehydrogenase enzymes (Δ(5)-3βHSD, 17βHSD2 and 17βHSD3). All of the tested compounds were effective in OH radical neutralization, particularly compounds 9, 11 and 14, which exhibited about 100-fold stronger activity than commercial antioxidants BHT and BHA. In DPPH radical scavenging new compounds were effective, but less than reference compounds. 2-Methoxybenzoyl ester 10 exhibited strong cytotoxicity against MDA-MB-231 cells. Most compounds inhibited growth of PC-3 cells, where salicyloyloxy stigmastane derivative 15 showed the best inhibition potency. Compounds 9, 10 and 11 were the best inhibitors of 17βHSD2 enzyme. X-ray structure analysis and molecular mechanics calculations (MMC) were performed for the best cytotoxic agents, compounds 10 and 15. A comparison of crystal and MMC structures of compounds 10 and 15 revealed that their molecules conformations are stable even after releasing of the influence of crystalline field and that the influence of crystal packing on molecular conformation is not predominant., (Copyright © 2014 Elsevier Inc. All rights reserved.)

New 17-picolyl and 17-picolinylidene androstane derivatives, 3-10, 15, 18, 19, 22 and 23, were synthesized starting from 17α-picolyl-androst-5-en-3β,17β-diol (1) and 17(Z)-picolinylidene-androst-5-en-3β-ol (2). Reaction of 1 with m-chloroperoxybenzoic acid gives 5α,6α-epoxy N-oxide derivative 3, or, with Jones reagent, 3,6-dione derivative 4; while 17α-picolyl-androst-5-en-3β,4α,17β-triol (5) or 3β,4β,17β-triol (6) derivatives are obtainable from 1 using SeO(2) in dioxane. Base-catalyzed tosyl group elimination from 7 or 9 affords AB conjugated derivatives 8 and 10. Oppenauer oxidation of 1 and 2 yields 4-en-3-one derivatives 11 and 12, which, with H(2)O(2) in 4 M NaOH, affords 4α,5α and 4β,5β-epoxides 13, 14, 16 and 17. New 4-methoxy-3-keto derivatives 15 and 18 were obtained from 13 and 14, or, with methanol in 4 M NaOH, from 16 and 17. Reduction of 11 with NaBH(4) gives 22, which was then acetylated to obtain 23. All new derivatives were screened for antitumor activity against human breast adenocarcinoma ER+, MCF-7; human breast adenocarcinoma ER-, MDA-MB-231; prostate cancer AR-, PC-3; human cervix carcinoma, HeLa; and colon cancer, HT-29 cells; as well as one human non-tumor cell line, MRC-5. Compounds 3, 5, 6, 8, 10, 18, 19 and 22 exhibited significant antitumor activity against MDA-MB-231 breast cancer cells; while 5, 6 and 10 also showed strong cytotoxicity against HT-29. Only compound 19 exhibited significant activity against MCF-7 breast cancer cells. No compounds displayed cytotoxicity against non-tumor MRC-5 cells., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)